Limited lineage relationship of tDCs and thymocytes revealed by retroviral barcoding. (A) Experimental setup. Fifty individually barcoded BaF3 cells were clonally expanded in vitro. Bar coded BaF3 clones were then mixed at defined ratios of 50:49:48 … 3:2:1 and 50 000 cells of these mixed cultures were subjected to bar code analysis by 454 sequencing. (B) Distribution of individual barcode sequences in BaF3 test sample. Expected frequencies of individual bar codes are shown in black. Observed frequencies of individual barcodes after sequencing are displayed in red. (C) Experimental setup to assess lineage relationship between tDCs, splenic DCs, and thymocytes. LSK cells were transduced with retroviral vectors encoding the barcode library at a multiplicity of infection, on average generating a single integration event per cell. Tagged LSK were FACS-sorted based on GFP expression and injected into lethally irradiated recipients to generate BM chimeras. About 8 to 10 weeks after transplantation, indicated populations of cells were sorted (50 000 cells) and sequenced as described in (A). (D) The distribution of unique barcodes was assessed for tDCs, splenic DCs, splenic B and T cells, DN, DP, and SP thymocytes, as well as BM lin⁻CD117⁺ precursors. Each color represents a unique barcode. (E) Unsupervised hierarchical clustering based on the MHI to reveal lineage relationships between sorted populations. (B,D-E) Data are pooled of 2 independent experiments with a total number of 6 mice analyzed.